The genomic DNA of all organisms undergoes spontaneous changes in the sequence (termed as mutation) in the course of their continuing evolution thereby generating variant forms of progenitor sequences, which may lead to various evolutionary advantages or disadvantages to the survival of the organism. If such effects of the mutations or variations are not seen then they are termed as neutral changes/mutations. If the mutation is lethal then it is not transmitted to the following generations and thus the mutation is lost from the gene pool of that organism. A variant form may also confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species, and hence, effectively it becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in the gene pool of the species. This coexistence. of multiple forms of a sequence gives rise to polymorphisms.
Several different types of polymorphism have been reported. A restriction fragment length polymorphism (RFLP) means a variation in DNA sequence that alters the length of a restriction fragment. The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses. Other polymorphisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetranucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analysis and in a large number of genetic mapping studies. Other polymorphisms take the form of single nucleotide variations. Such polymorphisms are far more frequent than RFLPS, STRs and VNTRs. Some single nucleotide polymorphisms (SNPs) occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Examples of genes, in which polymorphisms within coding sequences give rise to genetic disease include beta-globin (sickle cell anemia) and CFTR (cystic fibrosis). Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects.
The effects of such polymorphisms can be at various levels of cellular organization. Polymorphic elements in the promoter and/or regulatory regions are known to modulate the levels of mRNA of the genes. Polymorphisms in the un-translated regions (UTR's) of the RNA have also been documented to regulate the transcriptional and translational rates of the genes. Their presence in the intron-exon boundaries can also lead to changes in splicing and or splice products that are formed from the native full length mRNA. Polymorphisms in the coding region may change the function of the protein if it is a non-synonymous change and if it occurs in a critical domain of the protein leading to functional changes of the protein.
Thus polymorphisms are useful in defining genomic regions (for example as genetic markers) and they may also lead to disease (for example functional polymorphisms). Numerous examples are documented in the scientific literature and persons trained in this field are familiar with it (please see Abney M et al, Am J Hum Genet 70:920-34, 2002; Baron M, Mol Psychiatry 6:143-9, 2001; Bodmer W F, Ciba Found Symp 130:215-28, 1987; Breslow J L, Physiol Rev 68:85-132, 1988; Caraballo L R and Hernandez M, Tissue Antigens 35:182-6, 1990; Levitt R C, Am J Respir Crit Care Med 150:S94-9, 1994; Xu J et al, Clin Exp Allergy 28 Suppl 5:1-5; discussion 26-8, 1998).
Atopic diseases are a clinically heterogeneous group of diseases characterized by elevated serum IgE levels and varying phenotypic expressions such as Asthma and Atopic Dermatitis (Barnes K C, Clin Exp Allergy 29 Suppl 4:47-511999; Barnes P J Respir Res 2:64-5, 2001; Blumenthal M N and Amos D B, Chest 91:176S-184S, 1987; Thomas N S et al, Am J Respir Crit Care Med 156:S144-51, 1997). Specifically, Asthma is a chronic airway disease, affecting 15-18% of the world's population.
It is mainly a childhood disorder though the age. of onset can vary and is seen to be 35-45 yr. in the general population. Another case of extrinsic asthma is observed where the age of onset is above 45 yr. and is mainly due to the age induced changes in the lung function. The pathophysiology of atopic asthma is well documented. It is a T helper type 2 (Th2) mediated disorder with cytokines such as interleukin-4, interleukin-5, interleukin-13, implicated in the deviation of the immune system towards atopicity. Increased levels of these cytokines lead to elevated total serum IgE levels, eosinophil recruitment, and bronchial hyper-responsiveness that ultimately culminate in asthma pathogenesis. These interleukins are also known to interact and stimulate the alveolar cells and bronchial smooth muscle cells resulting in the clinical phenotypes of bronchial hyper-responsiveness (Barnes P J, Respir Res 2:64-5, 1999). Gene-gene and gene-environment interactions have been implicated in the development of asthma (Tay et al, Asian Pac J Allergy Immunol 17:239-42, 1999; Bleecker E R, Am J Respir Crit Care Med 156:S113-6, 1997; Cookson W, Nature 402:B5-11, 1999).
Various genetic studies have shown multiple loci to be associated with the disease. Asthma is therefore a multigenic disorder with a number of genes contributing minor effects leading to pathogenesis. Linkage studies, in various populations, have narrowed down the presence of susceptibility or disease genes to chromosomal locations such as 1p31, 5q31-33, 1p13, 12q13-24, 13q14, 17q12-21. However, all the causative genes and mutations have so far not been identified (Bleecker E R et al, Am J Respir Crit Care Med 156:S113-6, 1997; Blumenthal M N, Chest 91:176S-184S, 1987, Duffy D L, Epidemiol Rev 19:129-43, 1997).
Moreover, there is evidence to suggest that ethnic differences exist in the susceptibility genes associated with asthma (Xu J et al, Am J Hum Genet 68:1437-46, 2001). Of these loci, 12q21-23 harbors the Signal Transducer and Activator of Transcription-6 (STAT6) gene (consisting of 23 exons spanning a region of 19 kbp) which is thought to be an important candidate gene. STAT6 plays a major role in the initiation of signals from activated Th2 cells, specifically through IL4 and IL13 receptors (Ihle J N, Curr Opin Cell Biol 13:211-7, 2001; Zhu J et al, J Immunol 166:7276-81, 2001; Horvath C M, Trends Biochem Sci 25:496-502, 2000). STAT6 has also been implicated in the differential expression of chemokines, such as eotaxin-1, eotaxin-2 and thymus and activation regulated chemokine (TARC) (Takeda K and Akira S, Cytokine Growth Factor Rev 11:199-207, 2000; Zhang S et al, J Immunol 165:10-4, 2000; Mathew A et al, J Exp Med 193:1087-96, 2001). It is expressed in activated T cells in response to anti-CD3 antibody, PMA and other mitogenic responses (Arinobu Y et al, Biochem Biophys Res Commun 277:317-24, 2000). Interleukin 4 Receptor alpha (IL4RA) mediated phosphorylation of the STAT6 leads to its dimerization and nuclear localization, where it binds to the promoter elements of the Cεimmunoglobulin gene and causes the expression of the ε-transcript (Paul W E, Ciba Found Symp 204:208-16, discussion 216-9, 1997; Nelms K et al, Annu Rev Immunol 17:701-38, 1999; Linehan L A et al, J Immunol 161:302-10, 1998; Yang M et al, Am J Respir Cell Mol Biol 25:522-30, 2001).
Two naturally occurring isoforms have been detected that may modulate IL4 induced functional responses and cellular proliferation (Sherman M A et al, J Immunol 162:2703-8, 1999; Mullings R E et al, J Allergy Clin Immunol 108:832-8, 2001). The significance of this pathway in the development of atopic responses has been demonstrated by the failure of STAT6 (−/−) mice to develop a Th2 response, including, a lack in IgE production and eosinophilia, and failure to develop airway hyper-responsiveness in response to antigen challenge (Akimoto T et al, J Exp Med 187:1537-42, 1998; Miyata S et al, Clin Exp Allergy 29:114-23, 1999; Tomkinson A et al, Am J Respir Crit Care Med 160:1283-91, 2002; Zhu J et al, J Immunol 166:7276-81, 2001). A STAT6 antisense oligonucleotide was also shown to down regulate the expression of the germline ε transcript in DND39, a human Burkitt lymphoma cell line (Hill S et al, Am J Respir Cell Mol Biol 21:728-37, 1999).
Case control studies in the Japanese population have shown that a dinucleotide repeat in the 5′ UTR of this gene to be associated with asthma and atopic disorders (Gao P S et al, J Med Genet 37:380-2, 2000; Tamura K et al, Clin Exp Allergy 31:1509-14, 2001). However, they have not found any association of the repeat size with the total serum IgE levels. Also, this observation has not been confirmed in a more stringent study on a Caucasian sib pair cohort (Duetsch G et al, Hum Mol Genet 11:613-21, 2002). Duetsch et al has sequenced the complete gene and have identified a set of 23 SNPs spanning the intronic region. They have however not identified a polymorphism in the coding region. They were not able to demonstrate a significant association of these polymorphisms with asthma. These two studies suggest that there is a component of ethnic variation that is involved and that depends on the particular population under study.
In an earlier case control study in the Japanese population, the R3 locus has been found to be associated with asthma (13 repeat allele) (Tamura K et al, Clin Exp Allergy 31:1509-14, 2001). However, in a sib pair study in a German population, no such association of the R3 locus with asthma was seen, although weak associations were observed for the total serum IgE levels and the eosinophil counts with the alleles 17 and 16, respectively (Duetsch G et al , Hum Mol Genet 11:613-21, 2002). The present results of the present study provide very unique and unexpected results as shown in the prior arts. The association of allele 15 with asthma in the population could be explained are based not only on the ethnic differences that exist between observed in the present population and the Japanese and the Caucasian populations, but found generally in any population of the world. The present has identified the variants, which exist in any type of population in the world irrespective of its origin, community, colour, geographical location or ethnicity. The inventors have compared allele frequencies at R1 and R3 loci, and their haplotypes, in a population (comprising population from both North and South parts of India), they observed that their distributions are significantly different (data not shown). Also, the sampling strategies used in the studies are different. The present study is a case control study although the inventors have recruited individuals with a familial history of asthma and atopy. Further, the invention clearly defines that the variants identified would be useful for any kind of population of any geographical origin.
It is apparent that the use of the R1 and R3 polymorphisms in the generation of haplotypes in conjunction with SNP data for this gene may yield more informative haplotypes. The haplotypes of SNPs obtained in the German population suggests that there may be a recombination hot spot in the gene (Duetsch G et al, Hum Mol Genet 11:613-21, 2002). Estimation of decay of LD across the putative recombination hot spot could have been important in defining functional aspects of this genomic region. In any event, if functional polymorphisms are present on the chromosomal background of specific haplotypes then haplotypes that describe parts of the STAT6 gene flanking the putative recombination hot spot may provide a better association with asthma and total IgE. However, this hypothesis remains to be tested in the future.
Both the R1 and R3 polymorphisms seem to be biologically relevant. Using promoter deletion analysis it has been shown that the RI locus is flanked by the critical transcription factor binding sites TFIIIA and the TATA box (Patel B K et al, Genomics 52:192-200, 1998). Moreover, di-nucleotide repeats are known to bind various minor groove-binding proteins, which can interact with the basal transcriptional complex may modulate transcription. Interestingly, it has been shown that dinucleotide repeats have a propensity for forming Z-DNA like structures and that in the promoter regions these are capable of regulating transcription, for example, in the rat nucleolin gene (Rothenburg S et al, Proc Natl Acad Sci USA 98:8985-90, 2001). Also, CA repeats in the intron are known to regulate gene expression, for example in the first intron of epidermal growth factor receptor gene and interferon gamma genes (Gebhardt F et al, J Biol Chem 274:13176-80, 1999).
Similarly, the 5′-UTR is known to regulate translation of various genes through interaction with protein factors or by pseudoknot formation (Mokdad-Gargouri R et al, Nucleic Acids Res 29:1222-7, 2001; Ben-Asouli Y et al, Cell 108:221-32, 2002). However, further experimental work needs to be done to provide a conclusive proof for these hypotheses. In this context, it is important to note that, as shown by other groups and in the present study, no coding variants of STAT6 gene were found (Heinzmann A Clin Exp Allergy 30:1555-61, 2000, Duetsch G et al, Hum Mol Genet 11:613-21, 2002, Nagarkatti R and Ghosh B, 2002, in press). Thus it is possible that the action of STAT6 may be mediated mostly by the transcriptional and translation modulation of its levels, rather than due to structural changes in the protein itself. Thus, based on the above evidence it appears that STAT6 may be an important modifier locus that plays a significant role in regulating the atopic phenotypes depending on the ethnic background of the patients.